production of egg yolk immunoglobulin (IgY) against chimeric recombinant protein TCPA-CTXB-OMPW and investigating its protective effect on Y۱ cell line against CT and LT toxins

Background and Aim : Cholera is a severe diarrheal disease causes severe dehydration in infected persons with gram-negative bacteria called vibrio cholera. Cholera is still one of the main causes of death in children under ۵ years old. Applying of specific chicken egg yolk immunoglobulins (IgY) is an innovate technology to produce antibodies for passive immunotherapy and prophylaxis. In this study we produced egg yolk antibody (IgY) against a chimeric antigen from ۳ different proteins of OMPW-TCPA-CTB from vibrio cholera o۱. The inhibitory effect of on CT and LT toxins was investigated.Methods : The pET۲۸a plasmid containing the chimeric gene OmpW, ctxB and TcpA, was expressed in the E coli BL۲۱ (DE۳). The recombinant antigen was purified via Ni-NTA chromatographic column. After immunizing chickens with the recombinant protein, polyclonal antibodies (IgY) were purified from the egg yolk by water dilution method and analyzed on SDS-PAGE. The activity and specificity of the IgY antibody was analyzed by the indirect and the whole cell ELISA. Inhibitory effect of IgY against CT toxin was tested on the Y۱ cell line. Due to the structural and functional similarity of CT and LT toxins, the inhibitory effect of IgY produced against chimeric protein was also investigated on the inhibition of LT toxin.Results : Indirect ELISA data showed that injection of ۱۰۰ mg of protein for four times into the chicken produced a significant amount of IgY. CT toxin affects the Y۱ cells by changing the shape of the cell from elongated to round one and ۲۵۰micro gr of IgY in PBS inhibited the effect of this toxin. The inhibitory effect of IgY on LT toxin was ۱۰۰ micro gr.Conclusion : The chimeric antigen could stimulate the immune response effectively and increase the antibody titer significantly with four injection. In addition, the IgY produced against chimeric protein could effectively inhibit the effects of LT and CT toxins.

کلیدواژه ها:

CT toxin ، LT toxin ، vibrio cholera ، IgY Immunoglobulin ، Y۱ assay ، chimeric proteins